Department of Medicine, Emory University, Atlanta, GA 30322, USA.
Am J Physiol Gastrointest Liver Physiol. 2010 Sep;299(3):G687-96. doi: 10.1152/ajpgi.00527.2009. Epub 2010 Jun 17.
PepT1 is a di/tripeptide transporter highly expressed in the small intestine, but poorly or not expressed in the colon. However, during chronic inflammation, such as inflammatory bowel disease, PepT1 expression is induced in the colon. Commensal bacteria that colonize the human colon produce a large amount of di/tripeptides. To date, two bacterial peptides (N-formylmethionyl-leucyl-phenylalanine and muramyl dipeptide) have been identified as substrates of PepT1. We hypothesized that the proinflammatory tripeptide l-Ala-gamma-d-Glu-meso-DAP (Tri-DAP), a breakdown product of bacterial peptidoglycan, is transported into intestinal epithelial cells via PepT1. We found that uptake of glycine-sarcosine, a specific substrate of PepT1, in intestinal epithelial Caco2-BBE cells was inhibited by Tri-DAP in a dose-dependent manner. Tri-DAP induced activation of NF-kappaB and MAP kinases, consequently leading to production of the proinflammatory cytokine interleukin-8. Tri-DAP-induced inflammatory response in Caco2-BBE cells was significantly suppressed by silencing of PepT1 expression by using PepT1-shRNAs in a tetracycline-regulated expression (Tet-off) system. Colonic epithelial HT29-Cl.19A cells, which do not express PepT1 under basal condition, were mostly insensitive to Tri-DAP-induced inflammation. However, HT29-Cl.19A cells exhibited proinflammatory response to Tri-DAP upon stable transfection with a plasmid encoding PepT1. Accordingly, Tri-DAP significantly increased keratinocyte-derived chemokine production in colonic tissues from transgenic mice expressing PepT1 in intestinal epithelial cells. Finally, Tri-DAP induced a significant drop in intracellular pH in intestinal epithelial cells expressing PepT1, but not in cells that did not express PepT1. Our data collectively support the classification of Tri-DAP as a novel substrate of PepT1. Given that PepT1 is highly expressed in the colon during inflammation, PepT1-mediated Tri-DAP transport may occur more effectively during such conditions, further contributing to intestinal inflammation.
PepT1 是一种二肽/三肽转运体，在小肠中高度表达，但在结肠中表达不佳或不表达。然而，在慢性炎症（如炎症性肠病）期间，PepT1 在结肠中被诱导表达。定植于人类结肠的共生细菌会产生大量的二肽/三肽。迄今为止，已经鉴定出两种细菌肽（N-甲酰甲硫氨酸-亮氨酸-苯丙氨酸和 muramyl 二肽）为 PepT1 的底物。我们假设促炎三肽 l-Ala-gamma-d-Glu-meso-DAP（Tri-DAP），一种细菌肽聚糖的分解产物，通过 PepT1 转运进入肠道上皮细胞。我们发现，肠道上皮细胞 Caco2-BBE 中甘氨酰-丝氨酸（一种 PepT1 的特异性底物）的摄取被 Tri-DAP 以剂量依赖的方式抑制。Tri-DAP 诱导 NF-κB 和 MAP 激酶的激活，进而导致促炎细胞因子白细胞介素-8 的产生。在 Tet-off 系统中，使用 PepT1-shRNA 沉默 PepT1 表达可显著抑制 Tri-DAP 诱导的 Caco2-BBE 细胞中的炎症反应。在基础条件下不表达 PepT1 的结肠上皮 HT29-Cl.19A 细胞对 Tri-DAP 诱导的炎症大多不敏感。然而，在稳定转染编码 PepT1 的质粒后，HT29-Cl.19A 细胞对 Tri-DAP 表现出促炎反应。相应地，Tri-DAP 显著增加了在肠道上皮细胞中表达 PepT1 的转基因小鼠结肠组织中角质形成细胞衍生趋化因子的产生。最后，Tri-DAP 在表达 PepT1 的肠道上皮细胞中导致细胞内 pH 值显著下降，但在不表达 PepT1 的细胞中则没有。我们的数据共同支持将 Tri-DAP 归类为 PepT1 的新型底物。鉴于 PepT1 在炎症期间在结肠中高度表达，在这种情况下，PepT1 介导的 Tri-DAP 转运可能更有效，进一步导致肠道炎症。
